The gut-brain axis is bidirectional and the imbalance of the gut microbiota usually coexists with brain diseases, including Alzheimer’s disease (AD). Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a core lesion in AD and persistent ER stress promotes AD pathology and impairs cognition. However, whether the imbalance of the gut microbiota is involved in triggering the ER stress in the brain remains unknown.In the present study, fecal microbiota transplantation (FMT) was performed with gut microbiota from AD patients and APP/PS1 mice, respectively, resulting in two mouse models with dysregulated gut microbiota. The ER stress marker protein levels in the cerebral cortex were assessed using western blotting. The composition of the gut microbiota was assessed using 16S rRNA sequencing.Excessive ER stress was induced in the cerebral cortex of mice after FMT. Elevated ER stress marker proteins  were observed, which were rescued by 3,3-dimethyl-1-butanol (DMB). Notably, DMB is a compound that significantly attenuates serum trimethylamine-N-oxide (TMAO), a metabolite of the gut microbiota widely reported to affect cognition.The findings indicate that imbalance of the gut microbiota induces ER stress in the cerebral cortex, which may be mediated by TMAO. APT mice (Alzheimer’s disease (AD) is a neurodegenerative disorder that primarily involves the cerebral cortex and hippocampus . PatholoAPT mice . These pAPT mice . TrimethAPT mice . In prevAPT mice . CurrentIncreasing evidence indicates that chronic ER stress is highly correlated with cognitive function and sustained stress is considered a pathological driver of AD . EndoplaIn AD research, a variety of therapeutic approaches to reduce ER stress are under development and mostly include various small molecules that directly inhibit components of the UPR response, with significant efficacy in many preclinical models . HoweverAmpicillin, metronidazole, neomycin, and vancomycin hydrochloride were purchased from MedChemExpress . Antibodies against PERK, p-PERK, and p-eIF2α were purchased from Cell Signaling , and antibodies against eIF2α and β-tubulin were from Santa Cruz . The enhanced chemiluminescence (ECL) kit was purchased from Thermo Fisher Scientific . Mouse TMAO Elisa kit was purchased from Kisong Biotechnology . Hs-CRP Elisa kit was purchased from Elabscience Biotechnology , mouse LPS Elisa kit from Cusabio , and 3,3-dimethyl-1-butanol (DMB) from Sigma-Aldrich .Male, 6-week-old C57BL/6J mice were purchased from Shanghai Slac Laboratory Animal Center . The mice were given adequate food and water and housed in a laboratory with 24°C temperature, 50–70% humidity, and 12-h light/12-h dark cycles. All animal experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics and Welfare Committee of Shanghai Tenth People’s Hospital.n = 6) and the control group (n = 6). Mice in the ABX group were administered a sterile PBS solution containing an antibiotic cocktail  by gavage once a day for 3 days in a total volume of 0.2 mL. The control group was not subjected to any intervention. On the 4th day, three mice in each group were sacrificed and the remaining mice were sacrificed on the 17th day after eating a normal diet.Because an antibiotic cocktail was needed to empty the microbes from the mouse gut before FMT, the mice were divided into two groups to evaluate the potential effects of the antibiotic cocktail, the ABX group (n = 7) was administered the antibiotic cocktail by gavage for 3 days followed by gavage with PBS containing 20% sterile glycerol three times a week for 2 weeks. The ABX + FMT-APP/PS1 group (n = 7) was first administered the antibiotic cocktail for 3 days followed by FMT treatment by gavage for 2 weeks . The ABX + FMT-AD group (n = 7) was administered the antibiotic cocktail for 3 days followed by FMT treatment  for 2 weeks.To verify gut microbiota of AD patients and APP mice can cause ER stress in the cerebral cortex of wild-type (WT) mice, all animals were randomly divided into four groups: ABX group, ABX + PBS group, ABX + FMT-APP/PS1 group, and ABX + FMT-AD group. The ABX group was treated with an antibiotic cocktail for 3 days as described in the previous experiment . The ABXFurthermore, to verify whether ER stress in the cerebral cortex caused by gut microbial dysbiosis was associated with the gut microbial-associated metabolite TMAO, the mice were divided into six groups: ABX treatment, ABX + DMB treatment, ABX + FMT-APP/PS1, ABX + DMB + FMT-APP/PS1, ABX + FMT-AD, and ABX + DMB + FMT-AD. The duration and dose of the antibiotic cocktail and FMT treatment were the same as described above. DMB treatment was administered to mice from day 4 to day 17 of the experiment with drinking water containing 1% DMB and mice in the other groups were given normal drinking water.Alzheimer’s disease fecal donors were all from the cognitive impairment clinic of Shanghai Tenth People’s Hospital. A total of six AD fecal donors were selected. The mean age of the donors was 72.2  years (range 67–80); 50% were women, and 33.3% were APOE ε4 carriers. The mean MMSE score was 18.7 (SD: 1.2). Fresh feces of AD patients and APP/PS1 mice were collected, diluted with sterile PBS solution, vortexed, centrifuged as previously described, and a bacterial suspension was obtained . Then, tThe hs-CRP Elisa kit (Elabscience) was used to measure the serum hs-CRP concentration following the manufacturer’s guidelines, The mouse LPS Elisa kit (Cusabio) and mouse TMAO kit (Kisong) were used to measure LPS and TMAO in serum, respectively, according to the manufacturer’s protocol.Both cerebral hemispheres were removed from mice after cervical dislocation and stored at −80°C. The frozen cerebral cortex was homogenized in a pre-frozen RIPA buffer. The BCA kit was used to determine the protein concentration of the sample. Total protein (20 μg) was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with 5% BSA solution to inhibit non-specific binding. The membranes were incubated with the following antibodies: rabbit anti-p-PERK , rabbit anti-PERK , rabbit anti-p-eIF2α , mouse anti-eIF2α (1:500), and mouse anti-β-tubulin (1:500). Next, the membranes were incubated for 1 h with the appropriate secondary antibodies . The β-tubulin level was used as the loading control. ImageJ was used to determine the intensity of various molecular bands.PRJNA832124) to be released upon publication. QIIME 1 and the R package 3.5.12 were used for 16S rRNA sequencing analysis. The UniFrac distance metric and primary coordinate analysis were used for diversity analysis to evaluate the structural changes of the microbial communities in the samples (PCoA). LEfSe was used to identify a wide range of taxa. Heat-map was plotted using R package 3.5.1. The relative abundance of gut microbiota was analyzed by one-way ANOVA followed by post-hoc comparisons using LSD’s test for multiple groups’ comparisons.The QIAamp DNA Stool Mini Kit was used to extract microbial genomic DNA from fecal samples following the manufacturer’s instructions. Amplification and sequencing of the 16S rRNA gene were performed as previously described . The forpost-hoc test was used for data analysis with GraphPad Prism 9 software. Data were compared between the two groups using a two-tailed Student’s t-test. The data are presented as mean ± standard error of the mean (SEM) with a statistical significance of p < 0.05.In addition to the detailed description of the 16S rRNA data, one-way ANOVA followed by the least significant difference (LSD) pairwise comparison p > 0.999) and p-eIF2α/eIF2α  levels in the cerebral cortex (p = 0.655) and p-eIF2α/eIF2α  levels in the cerebral cortex  and p-eIF2α/eIF2α levels were increased . Similarly, in the ABX + FMT-AD group, p-perk/perk levels were increased  and p-eIF2α/eIF2α levels were increased . In contrast, p-perk/perk and p-eIF2α/eIF2α levels were not statistically different between the ABX and ABX + PBS groups  and hs-CRP  levels were not statistically different between the four groups  and diversity index (Shannon) showed no significant difference between the groups , indicatBacteroidetes  at the phylum level and S24-7  at the family level, and a significant increase in the RA of Firmicutes  at the phylum level and Lachnospiraceae , Desulfovibrionaceae , and Ruminococcaceae  at the family level. Furthermore, similar changes were observed in the ABX + FMT-AD group. At the phylum level, the RA of Bacteroidetes decreased  and the RA of Firmicutes increased . At the family level, the RA of S24-7 decreased  and the RA of Lachnospiraceae , and Desulfovibrionaceae  increased   has been reported in AD patients and mouse models with gut microbiota dysbiosis. In addition, TMAO can enter the blood and cross the blood-brain barrier. In an in cells . Therefo 0.0232) . These rP < 0.0001; ABX + FMT-AD vs. ABX + DMB + FMT-AD, P = 0.0029)  . And exc 0.0029) . Thus, gFirmicutes/Bacteroidetes ratio  may act as a key signaling mediator . In a reThere are some limitations to our study. First, our study used only male mice, so it can only reflect the relationship between gut microbiota dysregulation and ER stress in the male mouse population. Second, the cognitive function of the recipient mice and the pathology of AD in the brain, such as Aβ and p-tau, were not explored further. Therefore, further exploration of these issues is also the next step of our study. Third, extracellular bacterial DNA has been shown to play an important role in protein misfolding , yet theIn summary, the present study results indicate for the first time that host microbiota imbalance may lead to ER stress in the cerebral cortex and ER stress can be rescued by inhibiting TMAO production. In addition, an association was found between gut microbiota and the brain. These results can be used as a basis for reducing ER stress in the brain through modulation of the gut microbiota or its metabolites, contributing to the understanding and treatment of neurological disorders associated with ER dysfunction.PRJNA832124.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI–The studies involving human participants were reviewed and approved by Ethics Committee of Shanghai Tenth People’s Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Ethics and Welfare Committee of Shanghai Tenth People’s Hospital.XL and YZ designed the experiments and revised the manuscript. FW, CX, and YG performed fecal microbial transplantation and drafted the manuscript. FW performed the western blot experiments. KD and CZ performed the analysis of 16srDNA sequencing results. All authors agreed to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


GO-doping modification is promising for improving the performance and structure of TFC-FO membranes.Forward osmosis (FO) membranes have the advantages of low energy consumption, high water recovery rate, and low membrane pollution trend, and they have been widely studied in many fields. However, the internal concentration polarization (ICP) caused by the accumulation of solutes in the porous support layer will reduce permeation efficiency, which is currently unavoidable. In this paper, we doped Graphene oxide (GO) nanoparticles (50~150 nm) to a polyamide (PA) active layer and/or polysulfone (PSF) support layer, investigating the influence of GO on the morphology and properties of thin-film composite forward osmosis (TFC-FO) membranes. The results show that under the optimal doping amount, doping GO to the PA active layer and PSF support layer, respectively, is conducive to the formation of dense and uniform nano-scale water channels perpendicular to the membrane surface possessing a high salt rejection rate and low reverse solute flux without sacrificing high water flux. Moreover, the water channels formed by doping GO to the active layer possess preferable properties, which significantly improves the salt rejection and water permeability of the membrane, with a salt rejection rate higher than 99% and a water flux of 54.85 L·m In the past few decades, researchers have done many works to deal with water pollution and water shortage problems ,2,3. ForA good FO membrane material should have high salt rejection, good hydrophilicity, high water flux, high mechanical strength, and good stability, which can be affected by the active layer and support layer ,20,21. GGO-modified membranes have demonstrated good performance in FO processes ,31. ParkPSF/GO and TFC-FOPA/GO. Then, GO was simultaneously doped in the support layer and active layer, and such a membrane was represented as TFC-FOPSF-PA/GO. The GO loadings of the three membranes were equal. The morphological structures of the three TFC-FO membranes were characterized to explore the effects of GO on the membrane structure and properties. Finally, FO experiments were performed in pressure retarded osmotic (PRO) mode (active layer toward draw solution or AL-DS) and FO mode (active layer toward feed solution or AL-FS) to investigate the performance of different membranes.According to published results of GO-modified membranes, GO plays a crucial role in the final performance of the membrane. To deal with the agglomeration problem and prevent swelling of GO nanoparticles ,38, GO i6H12O6·H2O, analytical grade) for membrane permeability testing were supplied by Tianjin Komio Chemical Reagent Co., Ltd. .GO with a particle size of 50–150 nm was used as the membrane doping material. Polysulfone , N-methylpyrrolidone , N,N-dimethylformamide , n-hexane , trimesoyl chloride , and m-phenylenediamine  were supplied by Tianjin Yuanli Chemical Co., Ltd. . Sodium chloride  and glucose  was added to NMP solutions containing 8 wt% polysulfone. The prepared mixture was stirred in a water bath at 60 °C and stood for 24 h to defoam. With an automatic membrane coating machine, the membrane was coated on a glass plate to obtain a PSF/GO base membrane with a thickness of 100 μm.PSF/GO membrane. For TFC-FOPSF/GO-0, TFC-FOPSF/GO-0.05, TFC-FOPSF/GO-0.15, TFC-FOPSF/GO-0.2 and TFC-FOPSF/GO-0.3, GO nanoparticles were only added to the PSF support layer, and the loadings of GO (weight ratio to PSF) were 0 wt%, 0.05 wt%, 0.15 wt%, 0.2 wt%, and 0.3 wt%, respectively.An interfacial polymerization (IP) method was employed to prepare the dense active layer of the membrane . The prePA/GO membrane was prepared by the IP method. GO nanoparticles were first dispersed in a 2% MPD aqueous solution by ultrasound treatment. Then, the PSF base membrane was immersed in the prepared GO slurry with different GO contents  for 2 min. The subsequent preparation method was similar to the method used for TFC-FOPSF/GO preparation. For TFC-FOPA/GO-0.05, TFC-FOPA/GO-0.15, TFC-FOPA/GO-0.2 and TFC-FOPA/GO-0.3, GO nanoparticles were only introduced to the PA active layer, and the loadings of GO (weight ratio to PA) were 0.05 wt%, 0.15 wt%, 0.2 wt%, and 0.3 wt%, respectively.For the membrane support layer, a PSF membrane without GO doping was prepared. TFC-FOPSF-PA/GO-0.05, TFC-FOPSF-PA/GO-0.15, TFC-FOPSF-PA/GO-0.2 and TFC-FOPSFPA/GO-0.3 mean that GO nanoparticles were added to both the active layer and support layer, and the total GO loadings in the two layers were 0.05 wt%, 0.15 wt%, 0.2 wt%, and 0.3 wt%, respectively.The phase inversion method in −1. Field emission scanning electron microscopy  was used to observe the surface and cross-sectional morphology of fo films. First, the sample was completely dried. When observing the plane, cut the sample into appropriate sizes and paste it on the plane observation platform with conductive adhesive. When observing the section, the small sample chamber shall be brittlely broken with liquid nitrogen and pasted on the section observation platform with conductive adhesive. Next, gold spraying was performed with a gold spraying apparatus under the conditions of a gold spraying current of 15 mA and a gold spraying time of 80 s. Finally, it can be placed in SEM and observed at 5 kV.Attenuated total reflection-Fourier transform infrared spectroscopy  was used to analyze the oxygen-containing groups of GO, and a scanning electron microscope (SEM) was employed to observe the GO nanoparticle morphology. The changes in the 2 theta angle of GO were observed by XRD spectroscopy . The Fourier trans-form infrared spectrometer was used to identify Graphene Oxide over the wave number range of 500–4000 cmPSF/GO, TFC-FOPA/GO and TFC-FOPSF-PA/GO membranes. With a scan area of 5 × 5 µm, atomic force microscopy  was used to obtain the membrane surface roughness. The horizontal and vertical resolutions of AFM are 0.04 and 0.01 nm, respectively. First, the sample needs to be dried. Take a small piece of a flat sample, and paste it on the slide with double-sided adhesive tape. After that, the samples were tested using the tapping mode, and the surface topological junction of the fo film was obtained. The average roughness (Ra) and the root mean square roughness (Rq) of the film surface can be obtained through analysis of the supporting software. Moreover, we employed X-ray photoelectron spectroscopy  to analyze the membrane elemental composition, and optical tensiometer to investigate the surface hydrophilicity. The formation of the characteristic polyamide peaks was demonstrated with the aid of ATR-FTIR .Field emission scanning electron microscopy  at 15 kV was used to observe the surface morphology and microstructure of TFC-FOɛ) was analyzed using Equation (1). A dry sample with a mass of m2 was soaked in water for 24 h at 25 °C. After removing the residual water, we obtained the mass (m1) of the wet sample. The results of the test were the average of five measurements. The PSF/GO membrane porosity (R), reverse salt flux (Js), and water flux (Jv). The pure water permeation flux (Jv) under 2 bar of hydraulic pressure (ΔP) was measured using DI water as the feed solution. The flow length of the membrane tank was 60 cm with an effective membrane area of 1271 cm2, and the volume flow is 100 mL/min and the operation time is 1 h. While the temperature was fixed at 25 °C, pH = 7.5, the feed and draw fluids were circulated by two peristaltic pumps. For the Jv and Js analyses, DI water was used as the feed solution and 4.0 M NaCl as the draw solution. The extracted solution weight was continuously monitored using an electronic balance. For R analysis, we selected 0.5 M monohydrate of glucose (C6H12O6·H2O) as the draw solution and 1 mM NaCl as the feed solution. The calculations of Jv, Js, and R were conducted using Equations (2)–(4), respectively.Before evaluation, the FO membrane was immersed in deionized water at 25 ± 2 ℃ for 24 h. Using a laboratory-made forward osmosis test system, we tested the FO membrane salt rejection rate (m was the mass increase (g) of the draw solution, wρ was the density (g/L) of water, Δt was the run time (h), and mA was the effective membrane area (m2). tC and tV were the change of the feed solution concentration (mol·L−1) and the change of the draw solution volume (L), respectively. fC was the initial feed solution concentration (mol·L−1), and pC was the final draw solution concentration (mol·L−1).ΔThe XRD pattern of GO  showed tThe SEM characterization in PSF/GO, TFC-FOPA/GO and TFC-FOPSF-PA/GO were 100~400 nm, 300~500 nm, and 700~2000 nm. With the enlargement of the membrane pores, we could see that the membrane pores were intertwined with numerous dense reticular channels. This unique “network structure” provided high water flux and good salt interception performance of the membranes. For TFC-FOPSF/GO and TFC-FOPA/GO, the membrane water channels were dense and uniform after GO doping because the MPD-GO crosslinked body was crosslinked with TMC as well when the membrane contacted with TMC. However, the membrane water channels formed by doping GO in the two layers simultaneously were not fully developed, and the porosity was lower than that of TFC-FOPSF/GO and TFC-FOPA/GO. During the IP processes for the preparation of TFC-FOPSF-PA/GO membranes, the PSF-GO support layer was first reacted with the MPD-GO solution. The steric effect prevented GO nanoparticles from undergoing sufficient reactions with TMC. Therefore, more GO nanoparticles accumulated inside and at the edges of the membrane pores, which decreased the number of water channels and permeability.As shown in PSF/GO membranes had more water channels than TFC-FOPA/GO membranes. The active layer of TFC-FOPA/GO membrane was dense, which ensured a better salt rejection rate.In PSF/GO and TFC-FOPA/GO membranes had more obvious surface roughness compared with the TFC-FOPSF-PA/GO membrane as well as the membrane without GO doping (GO-0). The calculated surface roughness is listed in PSF/GO > TFC-FOPA/GO > GO-0 > TFC-FOPSF-PA/GO. The intra-membrane mobility of GO promoted the formation of porous support layers and dense water channels in TFC-FOPSF/GO and TFC-FOPA/GO membranes, which increased the roughness of the two membranes. When the support layer and active layer were doped with GO at the same time, the agglomeration of GO nanoparticles might have blocked the membrane pores and was not conducive to the effective formation of the porous structure, resulting in a reduction in roughness.The membrane surface roughness with the same doping amount was characterized by AFM . The TFCObviously, GO doping changed the surface morphology and microstructure of the TFC-FO membranes. In addition, GO contains a large number of -OH, -COOH, and other hydrophilic functional groups. After GO blending and modification, the membrane was more hydrophilic, while the interfacial reactions and polymerizations on the membrane surface were promoted accordingly.2) on MPD, but also with the oxygen-containing groups  on the GO surface to form amide and ester groups. The GO-involved reactions prevented the agglomeration of GO nanoparticles and generated water channels in the final FO membranes.We propose that the water channels in FO membranes are mainly generated in IP reaction processes. The possible reaction mechanism of the IP process used to prepare the active layers is illustrated in −1 and 1500 cm−1, which represented the stretching vibrations of the C = O and C-N bonds, respectively. The disappearance of -OH groups in the GO-doped TFC membrane was observed in the spectrum, which proved that GO participated in the reaction with MPD and TMC. The formation of polyamide groups promoted the formation of dense water channels. In most previous studies, the water channels in GO membranes were parallel to the membrane surface, while the GO-modified membranes studied in this paper possessed dense and uniform nano-water channels perpendicular to the membrane surface. Therefore, the proposed GO doping strategy could be used to modify the permeation performance of the membranes and reduce ICP.FTIR spectroscopy was employed to further prove the mechanism of the IP processes . The obvPSF/GO, TFC-FOPA/GO and TFC-FOPSF-PA/GO membranes. As depicted in the full XPS spectrum . The water channels formed by doping GO in the active layer possessed preferable properties. Doping 0.05 wt% GO in the active layer gave the FO membrane the highest


Alcohols and ethers are hard to oxidize, something which has a beneficial role in the anti-aged of recycled asphalt.With the continuous development of road construction and maintenance, SBS(Styrene-butadiene-styrene)-modified asphalt is widely used. However, there is no mature method for restoring aged SBS-modified asphalt. This study proposes the use of SBR(polymerized styrene butadiene rubber) and bio-oil for the restoration of aged SBS. In this study, five kinds of recycled asphalt were prepared by adding 5% bio-oil, 10% bio-oil, 6% SBR, 6% SBR + 5% bio-oil, and 6% SBR + 10% bio-oil to long-term aged SBS-modified asphalt. Softening point, penetration, and rotational viscosity experiments were tested to evaluate the conventional properties. Rheological tests revealed the performance of asphalt. Fourier transform infrared spectroscopy (FTIR), and atomic force microscope (AFM) tests were tested to demonstrate the microscopic characteristics of asphalt. Conventional tests investigated that aged asphalt viscosity will increase. Bio-oil could well recycle the asphalt viscosity. SBR could also soften aged asphalt, but its modification effect is limited compared with bio-oil. Rheological tests presented that the SBR and bio-oil have little impact on the temperature sensitivity of SBS-modified asphalt. SBR and bio-oil could decrease the asphalt stiffness. However, SBR and bio-oil could ameliorate the anti-cracking behavior of aged asphalt. The microscopic tests exhibited that SBR and bio-oil could decrease the asphaltene and colloid. Meanwhile, bio-oil could supplement alcohols and ethers at wave number 1000 cm With the development of road engineering and the increase of pavement maintenance mileage, asphalt usage increases yearly. As a by-product of petroleum, asphalt is a non-renewable material. With the extensive exploitation of oil resources, petroleum resources will be gradually depleted. At the same time, the demand for road construction and maintenance increased year by year, and the demand for asphalt also increased. During pavement maintenance, the waste aggregate and aged asphalt will pollute the environment . For greSBS-modified asphalt has excellent laboratory performance and durability, so it has been widely applied in the field of road engineering . In the A styrene-butadiene-styrene triple copolymer forms SBS; they create a gel network in the asphalt, strengthening its performance and obtaining good storage stability . On the The sulfoxide and aromatic hydrocarbon indexes of bio-oil recycled asphalt remarkably decreased, indicating that bio-oil had a better recovery function for aged asphalt . Lv 13]13] illusSBR is characterized by abrasion, heat, and aging resistance, such as SBS; it is a rubber material synthesized from butadiene and styrene as a unit. The cross-linking of rubber can further improve the ductility and anti-aged capability of modified asphalt . SBR-modIn summary, bio-oil was used in the regeneration of aged asphalt. Bio-oil could supplement the light components and enhance the low-temperature behavior of aged asphalt. SBR and SBS have styrene and butadiene copolymer materials, which ameliorate the low-temperature performance. SBR recycled asphalt can also play an excellent anti-fatigue effect; it can provide a new idea for recycled asphalt pavement (RAP). This study conducted laboratory tests on SBR/bio-oil composite regeneration aging SBS-modified asphalt to evaluate its behavior. FTIR and atomic force microscope (AFM) were tested to reveal their microscopic characteristics. This study provides the experimental basis for applying SBR/bio-oil regenerated SBS-modified asphalt.The task of regenerating the widely used SBS-modified asphalt is urgent in order to enhance the sustainable development of resources and the need for ecological and environmental protection ,31,32. ASBR and bio-oil were selected as the regenerants. The specific specifications and parameters of SBR are shown in Bio-oil produced by Nantong Yuhao Chemical Technology Co., LTD. . The specific parameters of bio-oil are shown in First, the SBS-modified asphalt (SBS) was subjected to short-term aging using a Rotating Film Oven (RTFO) at 163 °C for 85 min. Subsequently, the short-term aged asphalt was subjected to long-term aging in a Pressure Aging Vessel (PAV) to obtain SBS long-term aged, modified asphalt (SBS-PAV).The SBS-PAV was heated continuously for 1 h at 165 °C. First, we prepared the single regenerator-modified asphalt. SBR regenerated asphalt was prepared by blending 6%  of SBR into SBS-PAV. Bio-oil recycled asphalt was prepared by blending 5% and 10%  bio-oil into aged SBS-PAV. Next, the SBR was added to bio-oil recycled asphalt to prepare SBR/bio-oil composite recycled asphalt. The bio-oil and SBR contents are shown in Softening point, penetration, and viscosity were used to evaluate the conventional properties. Conventional tests ASTM D3461 , ASTM D5Temperature sweep (46–88 °C) and Frequency sweep (0.1–10 Hz) tests were conducted. Moreover, the rutting index (G*/sinδ) is used to evaluate the viscoelastic properties of the recycled asphalt. DSR specimens are made with 25 mm molds. The instrument model used in this study is the Anton-Paar MCR302 .SBS-PAV and SBS-PAV recycled asphalt were made into 101.6 mm × 12.7 mm × 6.4 mm standard specimens. BBR tests were conducted at −12 °C, −18 °C, −24 °C, and −30 °C. The bending creep stiffness (S) and creep rate (m) were used to describe the anti-creaking behavior of asphalt. According to ASTM D6648-08  specific−1. The instrument has a built-in middle and far infrared diamond ATR module. In this paper, an ATR accessory is used for infrared spectrum analysis.So as to study the change of functional groups in SBS-PAV caused by regenerant, SBS-PAV and recycled asphalt were performance the FTIR tests. FTIR results can display the functional groups of asphalt. The infrared spectrometer used in this study is Thermo Fisher Nicolet iS50 , with scanning wave numbers of 700–4000 cmThe atomic force microscope  uses a microprobe to approach the asphalt surface and generate an interaction force with the asphalt surface. The computer captures the micro force and draws an atomic resolution image of the asphalt surface so that the microsurface morphology and microstructure of the asphalt surface can be observed. The smoothness of the AFM specimen has a large influence on the test results. Therefore, we make the asphalt drops on the silicon sheet, and then the oven is heated for a moment to ensure the smooth surface of the specimen. The atomic force test was carried out at an ambient temperature of 25 °C. The instrument used in this study is Bruker Dimension Icon. This research observes and analyzes SBS, SBS-PAV, 5%bio, 6%SBR, 6%SBR + 5%bio.The penetrations of SBS and recycled asphalt are shown in The softening point of SBS and recycled asphalt are shown in The viscosity of SBS and recycled asphalt at 135 °C are shown in In this study, temperature sweep tests were conducted on SBS, 5%bio, 10%bio, 6%SBR, 6%SBR + 5%bio, and 6%SBR + 10%bio. The rutting index is an evaluation index to assess the high-temperature performance of different asphalts. The G*/sinδ value is shown in At 64 °C, the G*/sinδ with 5%bio was 8114 Pa. Moreover, G*/sinδ with 10%bio was 5972 Pa, which decreased by 26.4% compared with 5%bio. With the bio-oil content increase, the G*/sinδ process of recycled asphalt decreased, and 5%bio had better rutting resistance than that of 10%bio. However, the G*/sinδ of 6%SBR at 64 °C is 10393 Pa, which was much higher than SBS 4563 Pa and bio-oil recycled asphalt. 6%SBR has the highest resistance to rutting deformation. Compared with the recycling effect of bio-oil and SBR, SBR has less recovery ability to rutting index than that of bio-oil.The 6%SBR + 5%bio is 24.8% higher than SBS recycled asphalt’s rutting index, and its G*/sinδ is 5695 Pa. The combined effect of SBS and bio-oil is more obvious than that of bio-oil and SBR alone, and its G*/sinδ is lower than that of 6%SBR and 5%bio. From the index, it is the decline of resistance rutting ability, and from the recovery of asphalt, it is better for the recovery effect of aging asphalt.The G*/sinδ of 6%SBR + 10%bio is 52.2% lower than 6%SBR + 5%bio; its G*/sinδ is 2722 Pa. Under the joint action of SBR and bio-oil, its G*/sinδ is far lower than that of SBS. The rutting resistance of 6%SBR + 10%bio could not meet road engineering.Comparing SBR and bio-oil alone, SBR and bio-oil alone could restore the rutting resistance of aged asphalt to some extent. Under the coupling effect of SBR and bio-oil, composite restoring could restore the rutting index to a large extent, but the excessive amount of padding would make the asphalt not meet the requirement of road engineering.A frequency sweep test of SBS and recycled asphalt was performed at 70 °C and 76 °C, and the test results are shown in As shown in The rutting index of 6%SBR + 10%bio was inferior to SBS, and its high-temperature performance was reduced. However, the rutting index of 6%SBR + 5%bio was slightly higher than SBS, which indicates that 6% SBR and 5% bio-oil could basically restore the aging SBS to its original state. Among them, bio-oil could supplement the light components volatilized by asphalt during aging . HoweverTo study the low-temperature crack resistance of asphalt, BBR tests were carried out on SBS, SBS-PAV, and 6%SBR + 5%bio. The greater the stiffness modulus (S) obtained from the test. The stiffer and more brittle the asphalt is at low temperatures. The creep rate (m) reflects the rate at which the stiffness modulus changes with time. The larger m is, the better the asphalt’s dissipation capacity for temperature stress. At the same time, ASTM D6648 requires that the m value should be greater than 0.3 and the S value should be less than 300 MPa. In The SBS, SBS-PAV, 6%SBR, 5%bio, and 6%SBR + 5%bio were tested by the infrared spectrometer, and the aging mechanism and regeneration mechanism were analyzed from the perspective of chemical functional groups. −1 corresponds to the C=O stretching vibration of CO2, which may be caused by the CO2 in the air during the test [−1; 1690 cm−1 corresponds to C=C stretching vibration and C-O stretching vibration in olefins or aromatics; it is caused by the absorption of oxygen by unsaturated carbon chains, while C=C is mainly from SBS. The contraction of the peak at 1080 cm−1 is caused by the reaction of the elemental sulfur present in the asphalt with oxygen. Firstly, the increase of the polar covalent bond C-O and the decrease of the nonpolar covalent bond C-H lead to the increase of the bitumen viscosity. Second, SBS contains polybutadiene segments and polystyrene chains. Chain segments containing C=C aging accelerate the decomposition of SBS. The degradation of SBS is accelerated by aging, which indicates that aging has a greater effect on light components such as olefins or aromatics.By analyzing the FTIR spectra of SBS and SBS-PAV, we can find subtle differences in the chemical composition of the asphalt during long-term aging in the test . There i−1. There are multiple groups superimposed near the 1750 cm−1 wave number, including aldehyde groups, ester groups, carboxyl groups, and other groups. We first excluded aldehyde groups because they must have moderate peaks at 1400 and 1100 cm−1, and the peak at 1750 cm−1 must be a strong signal. Finally, the ester group was identified because 5%bio showed a new peak at 1160 cm−1, which belongs to the signal of the ester group vibration. The bio-oil produced a C=O stretching vibration at 1750 cm−1. A superposition peak appears at 2330 cm−1 with wave number attributed to C=O stretching vibration due to the incorporation of SBR. The absorption peak at wave number 1270 cm−1, specific to SBR, is the stretching vibration of the olefin C-H bond and carboxyl C-O bond. The absorption peaks at 1000–1270 cm−1 represent the C-O stretching vibration of ether and carboxyl groups. However, the others are not easily oxidized, which has a good effect on the aging resistance of recycled asphalt. The weak absorption peak near 960 cm−1 is caused by the stretching vibration of C-H. the absorption peak at 2330–2350 cm−1 corresponds to the C=O vibration of CO2, which may be caused by the action of CO2 in the air during the test [The SBS, SBS-PAV, 5%bio, 6%SBR, and 6%SBR + 5%bio morphologies observed by AFM scan are shown in 2 to 0.96 μm2. In this study, NanoScope Analysis software was used to analyze the average length and average area of the bee structures. The area of individual bee structures has increased by 134% after PAV aging, and the average increase in length of individual bee structures after PAV aging is 15% from 2.24 nm to 2.58 nm. The area increases from 0.81 μma and root-mean-square roughness Rq of the SBS as-built samples are greater than that of SBS-PAV. SBS-PAV roughness is 31% and 35% lower than the original SBS asphalt Ra and Rq, respectively. There is a correlation between lower roughness and more and larger bee structures. Thermal oxidative aging reduces the surface roughness of the asphalt and reduces the surface energy of the asphalt, which in turn has an effect on aggregate adhesion. The roughness Ra and Rq of 6%SBR + 5%bio are restored by 96% and 82%, respectively, relative to the original SBS asphalt. The above data reveal that SBR and bio-oil supplemented the lighter fraction of asphalt during the regeneration process, and the ratio of asphaltene to gum was reduced. Moreover, the light component has a restraint effect on the wax crystallization of the bee structure, and the peak of the bee structure is reduced, thus reducing the roughness.In DMT modulus is Young’s modulus calculated by fitting the force curve with the Derjaguin–Muller–Toropov model. SBS was regenerated and aged by mixing 6%SBR and different proportions (5% and 10%) of bio-oil. Conventional properties and rheological properties tests were used to reveal the properties of recycled asphalt. FTIR and AFM experiments were carried out to reveal the regeneration mechanism. The following are the main conclusions of this study.Under the combined action of SBR and bio-oil, the penetration of aged asphalt was restored. The workability of asphalt regenerated by bio-oil and SBR was improved. Under the effects of 6% SBR and 10% bio-oil, the penetration far exceeds that of SBS. At the same time, the viscosity was obviously reduced.DSR and BBR tests proved the rheological behavior of recycled asphalt. SBS asphalt after PAV had higher stiffness, and the recovery effect of SBR and bio-oil regenerated asphalt was not enough. Bio-oil has a better viscosity reduction effect and can enhance the anti-creaking behavior of asphalt, but it will weaken the asphalt’s high-temperature behavior. SBR has a general viscosity reduction effect. Adding SBR and bio-oil together in aged asphalt could play their advantages, and the regeneration effect is best.−1. These light components maintain good elasticity at low temperatures; they make the composite recycled asphalt of SBR and bio-oil have good anti-creaking behavior.AFM analysis has shown that bio-oil could soften asphalt, supplementing light components of SBS-PAV. SBR could restore the polymer in asphalt, thus increasing the adhesion and roughness of asphalt, positively impacting the fatigue resistance of the asphalt mixture. The new functional groups in recycled asphalt were known that bio-oil and SBR mainly supplemented with aromatic and polar functional groups at wave numbers of 1000–1270 cmIn this study, we have contributed to the restoration of SBS-modified asphalt aged with SBR and bio-oil composite by conventional, rheological and microscopic tests on the recycled asphalt. In the next step, we will investigate the effects of various bio-oils and their component differences on the macroscopic properties of recycled asphalt.


Botryococcus terribilis (B. terribilis), in vitro using hair follicle dermal papilla cells (HFDPCs). We isolated two types of cells from B. terribilis—green and orange cells, obtained from two different culture conditions. Microarray and real time-PCR results revealed that both cell types stimulated the expression of several pathways and genes associated with different aspect of the hair follicle cycle. Additionally, we demonstrated B. terribilis’ effect on collagen and keratin synthesis and inflammation reduction. We successfully isolated a novel compound, methylated-meijicoccene (me-meijicoccene), and C32 botryococcene from B. terribilis to validate their promising effects. Our study revealed that treatment with the two compounds had no cytotoxic effect on HFDPCs and significantly enhanced the gene expression levels of hair growth markers at low concentrations. Our study provides the first evidence of the underlying hair growth promoting effect of B. terribilis and its novel compound, me-meijicoccene, and C32 botryococcene.A person’s quality of life can be adversely affected by hair loss. Microalgae are widely recognized for their abundance and rich functional components. Here, we evaluated the hair growth effect of a green alga,  The immense psychological and sociological importance of hair contributes to an individual’s outlook . Hair loHair is the result of coordinated processes and signals leading to cellular proliferation and differentiation within the hair follicle (HF). The HF undergoes a cycle composed of traversing various phases—anagen (growth phase), catagen (degenerative phase), telogen (resting phase), and exogen (shedding phase). However, some hair cycle disturbances can interfere with the process, causing hair loss, including premature termination of anagen, the early onset of the catagen and telogen stages, and a prolonged telogen-to-anagen transition. These abnormalities can result in the miniaturization of hair shafts in the HF and/or an increase in the number of hairs in the catagen–telogen phase ,5,6,7,8.The dermal papilla is an important element in the study of the HF niche. It is home to a population of mesenchymal cells known as hair follicle dermal papilla cells (HFDPCs) and a reservoir of multipotent stem cells to replenish the niche . HFDPCs Botryococcus is a group of colonial freshwater microalgae that are known to accumulate hydrocarbons in the cells and the intercellular space [Botryococcus races: Race A, B, and L. Race A produces odd numbered n-alkadienes and trienes from C25 to C33, while race B produces C30 to C37 unsaturated hydrocarbons [40 isoprenoid hydrocarbon [B. braunii is the most studied species in the genus for biofuel production and is also attracting attention as a source for extracting functional substances such as carotenoids [B. braunii [B. braunii shows antioxidant activity in the livers, brains, and kidneys of rats [B. braunii in the skin was also investigated using different human skin cells and results showed that the extract reduces skin dehydration by stimulating stratum corneum hydration-related genes using keratinocytes, promotes collagen synthesis of dermal fibroblasts, and stimulates adipocyte differentiation [B. terribilis, a phylogenetically closely related species to B. braunii, has similar physiological and morphological properties. B. terribilis is sometimes compared with B. braunii in terms of hydrocarbon productivity, but little or nothing is known about the functional substances its cells might contain. More importantly, the use of this species in hair growth research has not been analyzed to date. The elucidation of its potential effect on hair growth will be of great value as the search for natural alternatives for hair growth agents is on the rise.Microalgae are photosynthetic organisms mostly found in the hydrosphere, widely spread from temperate to tropical zones. The success of microalgae and microalgae-derived substances as food, feed, fuel, and functional materials depends on thier high growth rate and undeniably rich bio-active compounds. Because microalgae are composed of a great variety of species, they come with many utilization possibilities with unknown potential ,12,13. Tar space . Based oocarbons . Race L rocarbon . Since hrocarbon . Howeverotenoids . In addi braunii . Moreove of rats . The effntiation . On the B. terribilis using HFDPCs and to understand its molecular mechanisms. We mainly focused on the changes in the gene expression levels of the pathways associated with hair growth and the possible difference of the effect of two types of B. terribilis cells. We performed global gene expression profiling using DNA microarrays to elucidate the stimulated gene sets contributing to the growth of the HF. In addition, we synthesized two compounds from B. terribilis, namely methylated-meijicoccene (me-meijicoccene) and C32 botryococcene. These pure compounds were used to further validate the effect of B. terribilis on hair growth promotion and to investigate their individual contributions to this effect.This study aims to evaluate the potential hair-growth-promoting effect of Botryococcus terribilis was isolated from the surface water of a reservoir located in Okinawa Island, Japan. The strain was isolated from the other microalgae and bacteria following the standard procedure of microalgal isolation [2 under continuous illumination with white LED with an intensity of 200 μmol/m2/s. “Green cells” were defined as cells that presented with a green color under the normal culture conditions described above; on the other hand, “orange cells” were cells that presented with an orange color after the change in the culture conditions under CO2 deficiency. The deficiency was achieved by introducing pure air without additional CO2, then green cells turned from green to orange over several days. After harvesting, both “green cells” and “orange cells” were freeze-dried and stored at −20 °C. A total of 100 mg of each sample was extracted using 70% ethanol at room temperature and placed in a dark enclosure for two weeks. Stock samples were filtered using a 0.22 μm filter unit. Green and orange cells will be referred to as BT-GC and BT-OC, respectively. Me-meijicoccene and C32 botryococcene were dissolved in DMSO and administered.The TEPMO-26 strain of solation . Culture2 flask . The cells were constantly monitored for media changes every two days, incubated under sterile conditions at 37 °C, and injected with 5% CO2 until it reached 80% confluency. Once confluent, the cells were passaged according to the culture guidelines of Cell Applications Inc. and were seeded into a new flask. HFDPCs were seeded onto a 96-well plate and 6-well plate with a density of 3 × 104 for further experiments. The cells were left to attach overnight then treated for 48 h.HFDPCs were purchased from Cell Application Inc., Tokyo, Japan. The HFDPCs used were derived from the normal human hair follicles of a 56-year-old Caucasian female’s temporal scalp. HFDPCs were cryopreserved at the second or third passage. The cells can be cultured up to six passages before going into senescence with a doubling time of 30 h and a viability of 95%. The HFDPCs were maintained using Papilla Growth Medium supplemented with fetal calf serum (FBS), insulin transferrin triiodothyronine, bovine pituitary extract, and cyproterone solution  in a 75 cm4 and left overnight to attach. Cells were treated with 1/2000 dilution of BT-GC and BT-OC, 0.1 μM minoxidil  as a positive control, and 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, or 10 μM of me-meijicoccene and C32 botryococcene for 48 h. After 48 h, the total RNA was extracted using ISOGEN kit  according to the manufacturer’s instructions and was quantified using the NanoDrop 2000 spectrophotometer .HFDPCs were seeded in a collagen-coated 6-well plate with a density of 3 × 10p-value ≤ 0.05 were considered differentially expressed genes (DEGs) and were used for further analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Visualization was partly conducted using R programming software.Template RNA was extracted using ISOGEN solution  as described above and was used to generate biotin-labeled aRNA and then purified and fragmented. The labeled and fragmented aRNA was subjected to hybridization using the GeneChip HG-U219 Array Strip Kit . The chip was washed and stained using the Gene Atlas Fluidics Station 400 and was scanned using the Gene Atlas Imaging Station . The resulting data were normalized using the Transcriptome Analysis Console (TAC) Software (version 4.0.1). Genes with fold change ≥ 1.5 (in linear space) and B. terribilis extracts, BT-GC and BT-OC, as well as the pure compounds, me-meijicoccene and C32 botryococcene, were subjected to gene expression analysis. The RNAs extracted as described above were used as a template for the RT-PCR analysis using Superscript IV VILO master mix  per the manufacturer’s protocol. TaqMan Universal Master Mix and TaqMan probes specific to β-catenin  , alkaline phosphatase  , fibroblast growth factor-1 , and Recombinant signal binding protein J   were used. The assay was performed using 7500 Fast Real-Time PCR Software 1.3.1  with GAPDH (Hs 02786624_g1)  as the endogenous control. All reactions were analyzed in triplicate.Based on the microarray results, the gene expression levels of hair growth-associated genes are analyzed for validation. The B. terribilis and were freeze-dried. Each sample was tested via the thin liquid chromatography (TLC) method, and the Rf values of 0.49 and 0.31 when using hexane as an eluent confirmed the presence of two main compounds, A and B. The Soxhlet extraction process is a conventional method for separating various carbohydrates from natural sources [m/z 609.2812) matrix on a JEOL JMS-700 instrument. IR spectra were performed in attenuated total refraction (ATR) mode on a Shimadzu FT-IR QATR-S.Two extracts, BT-GC and BT-OC, were prepared with 70% ethanol from carbohydrates produced by  sources . Soxhlet4 and were left overnight to attach. The cells were treated with different concentrations  for 48 h. After the treatment period, 5mg/mL MTT was added to the cells and incubated for four hours, followed by the addition of 10% sodium dodecyl sulfate (SDS) and incubation overnight. Absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as percentage (%) relative to the control or untreated cells.The cytotoxicity of me-meijicoccene and C32 botryococcene was determined by MTT assay. Briefly, HFDPCs were seeded in a collagen-coated 96-well plate with a density of 3 × 10p-value of ≤0.05 was considered significant. The results are the mean of three separate experiments.The results are expressed as mean ± SD. Statistical significance was determined by performing one-way ANOVA tests between the control and treated groups using GraphPad Prism 9 . A B. terribilis was cultured in a normal and CO2 deficient environment to yield two different types of cells: BT-GC and BT-OC. The study aims to differentiate whether there is a difference between BT-GC and BT-OC. p-value for each pathway. BT-GC and BT-OC enhanced several signaling pathways related to hair growth, including the PI3K-Akt signaling pathway that has 132 and 135 genes, the Ras signaling pathway that has 82 and 75 genes, the TNF signaling pathway that has 55 and 54 genes, the p53 signaling pathway that has 34 and 30 genes, the MAPK signaling pathway that has 101 and 96 genes, and the HIF-1 signaling pathway that consists of 39 and 40 genes. BT-OC enhanced the canonical Wnt signaling pathway that consisted of 52 genes and BT-GC enhanced 42 genes related to melanogenesis  and used as markers for the validation of hair growth effect. The gene expression analysis of BT-GC and BT-OC exhibited a highly significant increased expression level for CTNNB1 , ALPL , FGF1 , and RBPJ , respectively. The effect of the two studied samples on the gene expression of the validated makers was rather similar.  further enhance the hair growth activity of these samples in dermal papilla cells. (Lef and TCF1 are discovered to be positively stimulated by BT-GC and BT-OC (DKK1, a Wnt antagonist, was found to be negatively affected by BT-GC and BT-OC. The expression of DKK1 suppresses Wnt signaling to prevent the premature differentiation of neural precursor cells [B. terribilis.The stimulating effect of a cells. . Wnt/β-cnd BT-OC . Lef is or cells . The actNotch binds to RBPJ to impose its transactivation activities, which include hair differentiation [FGF1, FGF7, FGF12, and FGFR1 were listed. FGF1 stimulates dermal papilla proliferation, promotes the cell cycle, and facilitates hair regeneration. FGF7 acts on the promotion of the anagen phase and is responsible for HF morphogenesis and the regulation of the hair growth cycle. FGF12 was found to be associated with several pathways such as the MAPK pathway, Ras pathway, and PI3k-Akt pathway. [ALPL, a prominent dermal papilla growth marker [Other relevant pathways were amplified including Notch signaling, ERK1/2 signaling, and MAPK signaling A,C. In tntiation . The ERKntiation . These antiation ,39,40. Mpathway. ,42,43. Ah marker ,45. KEGGh marker . TNF sigh marker . Ras sigh marker .B. terribilis prolongs the early anagen phase by slowing down the cell cycle progression through blocking the transition of cells from the G1 to the S phase [The hair cycle is an important biological clock orchestrated by processes such as the cell cycle. In parallel to this, we observed the downregulated gene ontologies associated with aging, cell cycle arrest, and cell death B,D. Cell S phase , thus deB. terribilis. Thus, the suppression of the pro-inflammatory genes by B. terribilis strengthen the effect of these samples on stimulating hair growth and preventing hair shaft shredding [CTNNB1, ALPL, FGF1, and RBPJ. These four markers were chosen due to their important roles in hair growth. CTNNB1 is essential for anagen initiation, ALPL is widely known as a HFDPCs proliferation marker, FGF1 is involved in morphogenesis, and RBPJ is linked to the NOTCH pathway that regulates hair differentiation. Choosing these three markers allows us to cover different aspect of the HF cycle. The treatment of BT-GC and BT-OC strongly enhanced the gene expression levels of the t markers after 48 h of treatment (B. terribilis in vitro. Our findings suggests that B. terribilis has a significant effect on hair growth-associated genes, has a potential anti-inflammation effect, and can be an alternative treatment for hair loss.Androgenic alopecia and alopecia areata are characterized by a sustained follicular inflammation that leads to hair loss. A heatmap showing the relative expression intensity A revealshredding . Moreovehredding . The epihredding . Additiohredding . Due to reatment C,D. CurrB. terribilis described by our study, extracts are a very complicated mixture of various substances. To identify and purify the compound that may be the main driver of this effect, HPLC analysis was performed. We have successfully isolated C32 botryococcene and the novel compound me-meijicoccene found in B. terribilis. C32 botryococcene is a known compound; however, this is the first time its NMR data and characteristics have been reported. Me-meijicoccene is a novel compound and to our knowledge no study has reported this compound before. C32 botryococcene and me-meijicoccene did not affect HFDPCs’ viability (CTNNB1, ALPL, FGF1 and RBPJ significantly at a lower concentration (Despite the compelling evidence of the hair growth promotion effect of iability A. Additintration . TherefoB. terribilis extracts BT-GC and BT-OC exhibited a promising hair growth effect through the enhancement of the expression of important hair growth-related genes. B. terribilis’ stimulation of anagen markers, downregulation of catagen inducer genes, repression of Wnt inhibitors, and supplementation of the stimulation of other pathways contributed to the hair growth promotion effect. Overall, this study provides the first report of the effect of B. terribilis, as well as the novel compound me-meijicoccene and C32 botryococcene, on hair growth. Clinical studies are deemed necessary to validate the observed effect of B. terribilis, its novel compound me-meijicoccene, and C32 botryococcene.In conclusion, given the profound effect of hair loss on an individual’s quality of life, there is a need to explore alternative therapeutic agents for inducing hair growth. In parallel to this, there is a huge demand for nature-derived agents to fill this gap. Treatment with the The data reported in this article have been used to apply for a patent under Japanese Patent Application No. 2022-036924.


Spearman’s correlation coefficient results were significant but with a small effect size . In conclusion, the present results suggest that the PAQ-C-It can be cautiously used as tool to practically classify active Italian children because of a non-solid relationship between respective accelerometer data and MVPA daily data.The present study aimed to determine a cut-off value following the filling in of a questionnaire (PAQ-C-It) to identify active Italian children. One-hundred-twenty-nine primary school children  wore an accelerometer (Actigraph wGT3X-BT) to objectively quantify individual moderate-to-vigorous physical activity during one week. Afterwards, the PAQ-C-It was filled in by participants. A ROC curve procedure was applied to obtain an active/non-active cut-off point. Spearman’s correlation coefficient was also applied to establish the relationship between the two parameters. According to the ROC analysis, the PAQ-C-It cut-off point value is identifiable at >2.75 to indicate active children (area under the curve = 0.62; standard error = 0.05;  Physical activity (PA) is considered a key predictor of physical and mental health in children and adolescents ,2. On thIn order to monitor and assess the adherence to MVPA, as well as PA in general, different measurement methods, both subjective  and objective  are applied ,15. AmonAmong self-report methods, PA questionnaires are practical to administer, cost-effective and allow an insight into the PA of a large-scale population . In partFurthermore, when validating concurrent validity, the new assessment should be compared to a well-established measure. Nonetheless, the calibration process of the accelerometers through the definition of count thresholds and equations to process raw information led to a high variability in comparing the results of different research studies ,34. MoreHowever, cut-offs for MVPA should be defined in accordance with specific populations. In fact, PA in children varies in relation to several demographic, biological, environmental, social and psychological factors . Therefon = 23) were excluded. A final sample of 126 children (47.6% girls) participated in this study.An invitation to participate in the study was sent to all parents who had their children and adolescents in five primary schools in the neighborhoods of Turin . Sessions to provide preliminary information regarding the aim of the study to the children’s teachers and parents/guardians were held in all recruited schools. All schools/classes recruited for the study were part of the same institutional and regional office and therefore were receiving a similar delivery of educational curricula . Parents−2).Stature was measured using a portable stadiometer  with an accuracy of 0.01 m. Body mass was measured using an electronic scale  with an accuracy of 0.1 kg. Both stature and body mass were measured without shoes. Waist circumference was measured to the nearest 0.01 m midway between the lowest rib and the iliac crest with the participants in the standing position. The Body Mass Index (BMI) was calculated as body mass divided by height squared  was used to assess PA objectively. The GT3X-BT is lightweight (27 g), compact (3.8 × 3.7 × 1.8 cm), and has a rechargeable lithium polymer battery. In this study, accelerometer data was sampled at 30 Hz and summarized over 10 s epochs. The GT3X-BT proved to be a reliable and valid tool for the assessment of different types of physical activities, despite the accelerometer being word on the hips . Each paActilife Software (version 6.13.4 Actigraph Corporation) was used to process the accelerometer data. Periods of ≥60 min of zero values, allowing for 2 min of non-zero interruptions, were defined as accelerometer “non-wear” time and were removed from the analyses. The first day of recording  as well as the last one  were not included in the analysis. Only participants with ≥4 complete days, including one weekend day, were included . A day wus study , we consSubjective PA was assessed using the PAQ-C-It  at the ep < 0.05.Extreme outliers for outcome variables, defined as above or below 2 standard deviations from the mean, were identified and excluded from the analyses. Participants’ anthropometric characteristics and PAQ-C-It and MVPA were described in terms of means and standard deviations (SDs). Independent t-tests were conducted to determine whether there were statistically significant differences in anthropometric characteristics and outcome variables. The relationship between the PAQ-C-It and minutes of MVPA was investigated using Spearman’s rank correlation coefficients (rho). Correlations < 0.29 were considered weak, between 0.30 and 0.39 moderate, between 0.40 and 0.69, and those above 0.70 were considered “very strong” . FinallyApproximately 15.4% of eligible students were included in the study. The characteristics of the entire sample and considering gender are described as means and standard deviations (SDs) in p = 0.02; ES = 0.41; 95% CI = 0.06–0.77). A weak correlation  was found between the PAQ-C-It and minutes of MVPA. The plot of the ROC curve and the PAQ-C-It ROC curve coordinates  are reported in Briefly, no differences in terms of anthropometric characteristics were observed. Only for MVPA did females show a lower score than their gender counterparts  and 1-specificity ) with an associated AUC of 0.62 (95% CI: 0.52–0.72) and Youden index of 0.21. Based on the AUC, the classification accuracy was poor. The specificity value shows the low capacity of the PAQ-C-It to identify non-active children. For more details about statistical analysis, see To distinguish between active and non-active individuals, PA cut-offs should be defined in accordance with several factors associated to the specific population . TherefoTo our knowledge, this is the first study able to significantly define a PAQ-C-It cut-point value by accelerometry based on PA recommendations and with a device worn on the wrist. In fact, although our results reported a weak correlation between MVPA accelerometer and PAQ-C scores, this index could be considered useful for contributing to a better understanding of PAQ-C values. In addition, the ROC analyses reported a 2.75 PAQ-C-It cut-off score as a value to distinguish between Italian non-active and active children, which is the same threshold shown for co-aged Spanish children , in whicp < 0.05) for assessing MVPA in Italian children.Although the present study, as well as the previous one with Spanish children of a similar age (1 year of difference), reported low sensibility and specificity values, and a poor AUC. The latter value for Italian children is significant and closer to the “fair” threshold. Nevertheless, these results should be considered with caution because the AUC should be >0.7 for an acceptable clinical. In fact, even though the PAQ-C-It is not associated with a clinical context, but with a public health one, it has anyway been associated with a poor validity (rho = 0.21, From a methodological point of view, we considered the cut-off points for non-active children according to the calibration procedure described in a previous study specifically focused on children of 8–12 years old who mounted the accelerometer on their non-dominant wrist . TherefoThe children who participated in the present study reported homogenous age, anthropometrics, and PAQ-C-It values between girls and boys, but a different mean MVPA level, where girls showed higher values. Besides a controversial trend with respect to previous studies ,50, the Although the PA of the Italian children recruited in the present study can be classified as “moderate activity” according to the questionnaire scores for both genders ,36,37, tThe present study was able to demonstrate that the PAQ-C-It can be used only with caution as tool to practically classify active Italian children because of a non-solid relationship between respective accelerometer data and MVPA daily data. As a consequence, it could be partially considered only for those cases in which it is not possible to measure children’s PA by means of an accelerometer or other objective tools.


After treatment, OSDI, FL, FBUT and SIT were all obviously restored in the two groups. When comparing the changing rates, only OSDI  and FL  showed significant differences between the two groups. This study suggests that OSDI is not applicable in the diagnosis of dry eye among blepharospasm patients. For blepharospasm patients with severe dry eye symptoms, especially those with fluorescein staining in the cornea, the combined treatment of BoNT-A and APP is more effective than using BoNT-A alone.Blepharospasm patients often have dry eye manifestations. Botulinum neurotoxin type A (BoNT-A) injection has been the main management for blepharospasm and absorbable punctal plug (APP) insertion is shown to be effective in the treatment of dry eye. However, there have been no studies investigating the combined treatment of BoNT-A and APP in blepharospasm patients with dry eye. In this retrospective study, 17 blepharospasm patients with dry eye treated by BoNT-A injection and 12 receiving BoNT-A plus APP treatment were enrolled. The efficacy was evaluated according to the Jankovic rating scale, Ocular Surface Disease Index (OSDI), fluorescein staining (FL), fluorescein tear break-up time (FBUT) and Schirmer I test (SIT). Both BoNT-A and BoNT-A+APP treatment effectively reduced the functional impairment of blepharospasm. At baseline, all the patients had high OSDI scores (BoNT-A group: 82.48 ± 7.37, BoNT-A+APP group: 78.82 ± 4.60,  Blepharospasm, a subtype of focal dystonia, is often manifested as the abnormal contraction of the orbicularis oculi muscle ,2. BenigAs reported, blepharospasm and dry eye commonly occur together ,11. Dry This study aims to evaluate the clinical effectiveness of the combined treatment of BoNT-A injection and APP insertion on blepharospasm patients with dry eye. We compared the symptoms and signs associated with blepharospasm and dry eye in patients managed by BoNT-A+APP and in those treated by BoNT-A only. The changing rates of dry eye symptoms and signs before and after treatment were also investigated.This retrospective consecutive cohort study was conducted at Union Hospital, Huazhong University of Sciences and Technology, China. The patients were retrospectively recruited from 1 January 2020 to 31 May 2021. The study adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committee of Union Hospital, Huazhong University of Sciences and Technology.Twenty-nine adults diagnosed with blepharospasm with typical dry eye manifestations but failing to respond to ocular medications were consecutively enrolled. All the patients were administered the standardized dry eye treatment using artificial tears  three times a day before, but did not show any improvement in the dry eye symptoms. Twelve patients receiving the combined treatment of BoNT-A and APP were assigned to the observation group. Seventeen patients undergoing BoNT-A injection alone served as the control group. The diagnosis of blepharospasm was made in line with the published standard criteria . SubjectThe symptoms of blepharospasm were clinically graded according to the Jankovic rating scale. This scale includes two subscales that measure the severity and frequency of eyelid spasms, in each case on a 5-point scale ranging from 0 to 4 .A validated Chinese-translated version of the Ocular Surface Disease Index (OSDI) questionnaire was used for the assessment of dry eye symptoms. The overall OSDI score was determined as suggested in a previous study and defined the dry eye symptoms as mild (13–22), moderate (23–32) or severe (≥33) .After completing the questionnaire, the participants underwent a rigorous ophthalmologic investigation by the same specialist of the anterior segment. Firstly, fluorescein staining (FL) was conducted after dropping 2% sterile fluorescein sodium staining solution into the conjunctival sac and the examination was conducted for 2–3 min counting from the last instillation by passing light through a cobalt-blue filter and examined via a slit lamp. The classification of the entire corneal surface was facilitated by lifting the upper eyelid. The severity of corneal punctate dots was recorded using Oxford Schema: 0–5 of staining ranges for each panel  and 0–15 for the entire exposed inter-palpebral conjunctiva and cornea .In the detection of the fluorescein tear break-up time (FBUT), patients were asked not to blink after fluorescein instillation. The length of time after a patient blinked to the introduction of dryness in the tear film was calculated in seconds. The evaluation was repeated three times, and the mean value was enrolled .We performed Schirmer I test (SIT) without anesthesia to quantify the production of tears. A filter paper (5 × 35 mm) was placed on the lower conjunctiva within the temporal one-third area. After 5 min, the length traveled by the tears on the filter paper was measured. SIT was indicative of a water-deficient state if the measured distance was less than 5mm .All patients in this study were evaluated with Jankovic grading, OSDI, FL, FBUT and SIT before and after BoNT-A injections or BoNT-A+APP treatment. The changing rates ((after-before)/before) of each index were compared in the two groups.The BoNT-A injection preparation was made by mixing 100 units of purified dry botulinum toxin  with a 2.5 mL of saline solution. In the periocular region, the injection of BoNT-A was performed subcutaneously at 17 selected sites and the dose was 2 or 4 units per small or big dot injection site . The staFor patients receiving BoNT-A+APP combination treatment, APP  was used to occlude the upper and lower lacrimal puncta in both eyes after BoNT-A injection.After BoNT-A or BoNT-A+APP management, there was no specific treatment in those patients except using sodium hyaluronate eye drops three times a day if the patient needed it. The patients were followed up regularly on the next day, the first week and 4–6 weeks after BoNT-A injection or BoNT-A+APP treatment, and the clinical features were recorded before treatment, one week after and at the last visit.t-test was used for statistical comparisons between the BoNT-A+APP group and the BoNT-A group. A paired-samples t-test was applied to compare the efficacy before and after treatment in the same group. We considered a p-value inferior to 0.05 as significant statistical.For all variables in this study, the arithmetic mean ± standard deviation data were calculated. An independent-samples A total of 29 right eyes from 29 patients with blepharospasm and typical dry eye manifestations were included in the study. The demographic characteristics of the patients were almost the same among the two groups. In this study, 64.5% (11/17) of patients in the BoNT-A group and 75% (9/12) in the BoNT-A+APP group were female. Most of the patients were over 60 years old and the median ages were 67 (58.5–73.0) and 64 (59.3–67.0) years in the BoNT-A group and the BoNT-A+APP group , respectp = 0.586 in severity and p = 0.452 in frequency). After treatment, the severity and frequency of blepharospasm improved perceptibly in both the BoNT-A group and the BoNT-A+APP group  in the severity and frequency of blepharospasm. Most of them reported functional blindness and had trouble performing daily life activities. No significant differences were observed between the BoNT-A group and the BoNT-A+APP group before treatment (PP group .p = 0.112). However, dry eye signs were relatively weaker according to the results of FL , FBUT  and SIT . In the BoNT-A group, OSDI (p = 0.000) and FL (p = 0.003) were apparently decreased after treatment, with FBUT (p = 0.000) and SIT (p = 0.001) significantly increased (p = 0.000, p = 0.000, p = 0.015, p = 0.000, For the symptoms and signs of dry eye at baseline, no significant differences were found in OSDI, FL, FBUT and SIT between the patients treated by BoNT-A and BoNT-A+APP. Before treatment, all patients in the two groups had severe dry eye symptoms based on the high OSDI scores (BoNT-A group: 82.48 ± 7.37, BoNT-A+APP group: 78.82 ± 4.60, ncreased . This inp = 0.047, p = 0.016, p = 0.968, p = 0.270, Furthermore, to determine whether the combined management of BoNT-A and APP have a joint effect on the improvement of dry eye in blepharospasm patients, we compared the changing rates of OSDI, FL, FBUT and SIT between the BoNT-A group and the BoNT-A+APP group after 4–6 weeks of treatment. Compared with the BoNT-A group, evident improvements were observed in the reduction rates of OSDI (BoNT-A group: −52.23% ± 15.57%, BoNT-A+APP group: −61.84% ± 9.10%, Clinically, blepharospasm patients have often been found to have dry eye manifestations, typically with a Schirmer test lower than 5 mm in 60% to 80% of cases . RegardiIn this study, we found a gender bias as 64.5% of patients in the BoNT-A group and 75% in the BoNT-A+APP group were female. This bias was consistent with previous studies. According to a retrospective study in Taiwan, a male/female ratio of 1.71 was observed in the incidence of blepharospasm and females had a significantly higher mean annual incidence than males in all age groups . AnotherBoNT-A has been used to manage a wide variety of illnesses, including blepharospasm, strabismus, nystagmus, protective ptosis, cervical dystonia, eyelid retraction, hyperhidrosis, cerebral palsy and chronic migraine ,31,32. BPrevious studies have confirmed the effect of BoNT-A injections in improving dry eye symptoms ,15. The The punctal plug has been tested as a relatively effective, safe and reversible method of preserving artificial tears and the aqueous layer on the ocular surface in treating dry eye . An APP Based on the positive effect of BoNT-A and APP on curing dry eye, we expected a joint effect of the combination of BoNT-A and APP in alleviating the dry eye symptoms in blepharospasm patients. Our study showed that BoNT-A plus APP insertion is better than only using BoNT-A in treating dry eye among blepharospasm patients. Compared with the BoNT-A group, the reductions in OSDI and FL in the BoNT-A+APP group were found to be more significant at the last follow-up visit, suggesting a superior improvement following the combined treatment of BoNT-A and APP. Indeed, our results prove the clinical efficacy of BoNT-A plus APP in the management of dry eye in blepharospasm patients. This treatment modality not only helped to restore the functional impairment of blepharospasm, including the severity and frequency of eyelid spasms, but also to reduce dry eye symptoms and corneal fluorescein staining. Therefore, we recommend a combination therapy of BoNT-A and APP in blepharospasm patients with severe dry eye symptoms, especially those who are found to have fluorescein staining in cornea.In conclusion, this study suggests that OSDI is not applicable in the diagnosis of dry eye among blepharospasm patients. For blepharospasm patients with severe dry eye symptoms, especially those with fluorescein staining in cornea, the combined treatment of BoNT-A and APP is shown to be more effective compared with using BoNT-A alone. This study provides a feasible treatment option for blepharospasm patients with dry eye.


In the mature brain, structural and functional ‘fingerprints’ of brain connectivity can be used to identify the uniqueness of an individual. However, whether the characteristics that make a given brain distinguishable from others already exist at birth remains unknown. Here, we used neuroimaging data from the developing Human Connectome Project (dHCP) of preterm born neonates who were scanned twice during the perinatal period to assess the developing brain fingerprint. We found that 62% of the participants could be identified based on the congruence of the later structural connectome to the initial connectivity matrix derived from the earlier timepoint. In contrast, similarity between functional connectomes of the same subject at different time points was low. Only 10% of the participants showed greater self-similarity in comparison to self-to-other-similarity for the functional connectome. These results suggest that structural connectivity is more stable in early life and can represent a potential connectome fingerprint of the individual: a relatively stable structural connectome appears to support a changing functional connectome at a time when neonates must rapidly acquire new skills to adapt to their new environment.  •The brain structural connectome can be used to identify an individual at birth•Functional connectivity is less stable and cannot be used as a fingerprint•Age at first scan and days between scans impacts on similarity•Structural and functional similarity appear closest in central sulcus•Age at first scan significantly impacts on parietal functional similarity This has enabled characterisation of connectomes delineating the structural and functional organisation of the brain at a macro-scale. These connectomes can be represented as large-scale matrices where each row and column correspond to brain subunit indices which may be structural or functional nodes, so that each matrix element describes ‘connectivity’ between two neural parts . The infThe developing brain is governed by dynamic processes that transform an amalgam of a few cells in early gestation into a complex organ capable of rapidly processing and integrating information. The foetal period is marked by cortical migratory processes, with anomalies at this stage frequently leading to neuronal migration disorders and atypical brain structure . A hallmUseful information about the perinatal brain and its subsequent maturation has already been acquired. In the perinatal brain, in parallel with structural changes, cortical neurons fire specific activity patterns which further regulate genetic expression and refine structure into functional systems . Highly Here, we investigate whether a structural and/or functional fingerprint is already established perinatally, by assessing the similarity of the structural and functional connectome of preterm born infants, who were scanned soon after birth and then again at term equivalent age. Unlike adults, the maturing brain is highly dynamic and undergoing rapid reorganisation. Therefore, we hypothesised that connectome similarity would be lowest when the time between scans was longest. In addition, brain activity becomes experience-driven during this early postnatal period and experiences increase daily as the infant interacts with the world ex-utero . Thus, w22.1Research participants were prospectively recruited as part of the developing Human Connectome Project (dHCP), an observational, cross-sectional Open Science programme approved by the UK National Research Ethics Authority (14/LO/1169). Written consent was obtained from all participating families prior to imaging.As part of the dHCP, a total of 63 subjects were scanned twice and had both functional and diffusion MRI data acquired. After pre-processing, 18 diffusion datasets were discarded due to poor registration to template space, resulting in a total of 45 subjects  with good quality diffusion MRI data. All infants were born preterm at a median of 32.29 weeks gestational age (GA) [range: 25.57–37], with their first scan acquired at a median of 35 weeks post menstrual age (PMA) [range: 29.29–37.43] and the second acquired at term equivalent age at a median of 41 weeks PMA [range: 38.43–44.86]. From the functional data, 18 subjects were discarded due to poor registration to template space and additional 14 had to be discarded due to significant signal loss during acquisition. Thus, there was a total of 31 subjects  with good quality functional connectome data, born at a median of 34.57 weeks GA [range: 27.57–37.00], had their first scan at a median of 35.57 weeks PMA [range: 30.86–37.43] and had their second scan at a median of 40.57 weeks PMA [range: 38.86–44.57]. The final sub-group with both good quality structural and functional data consisted of 26 subjects , born at a median age of 34.14 weeks [range: 28.71–37.00], with their first scan acquired at a median of 35.43 weeks PMA [range: 31.43–37.43] and the second acquired at a median of 40.93 weeks PMA [range: 38.86–44.86] (details described in 2.2All subjects underwent Magnetic Resonance Imaging (MRI) scanning at the Evelina Newborn Imaging Centre, St Thomas’ Hospital, London, UK. Structural, diffusion and functional data was acquired using a 3 Tesla Philips Achieva system  with customised neonatal imaging system including a 32-channel phased-array head coil  . Infants2, 1000 s/mm2 and 2600 s/mm2 spherically distributed in 64, 88 and 128 directions respectively, with 20 b = 0 s/mm2 images and parameters: Multiband factor 4, SENSE factor 1.2, partial Fourier 0.86, acquired in-plane resolution 1.5 × 1.5 mm, 3 mm slices with 1.5 mm overlap, repetition time (TR)/ echo time (TE) of 3800/90 ms and 4 phase-encoding directions  was calculated to characterise motion in each time point . In addi2.42.4.1To identify the optimal network density threshold for structural and functional connectome similarity, we tested the percentage of correctly identified subjects for all possible thresholds . Based oFor visualisation purposes, all similarity values were normalised by scaling relative to the maximum correlation between time-point 1 and all other subjects at time-point 2 dividing by the maximum value in each row . As shown in The same analysis was repeated for a sub-group of participants that had both structural and functional connectome data. This resulted in two 26 × 26 matrices containing structural and functional data, characterising the identifiability rate for the two modalities in the same individuals. The diagonal values of these matrices were extracted to provide median and range of structural and functional self-similarity.2.4.2In order to assess the effect of age in the structural and functional fingerprint, we first calculated the partial correlation between PMA at scan at time-point 1 and self-similarity (controlling for days between scans), and then the partial correlation between days between scans and self-similarity (controlling for PMA at time-point 1) for the whole group in each modality. Then, for the sub-group we converted the self-similarity and the self-to-other-similarity into z-scores for better visualisation of the effect of age on self-similarity. If the self-similarity z-score of a subject was higher than any of the self-to-other-similarity z-score, this would be equivalent to successfully matching a subject between time-point 1 and time-point 2, as in previous fingerprinting studies .To further characterise the effect of age on self-similarity, we ran a general linear model analysis with PMA at time-point 1 and days between scans as independent variables and self-similarity as dependent variable. This allowed quantification of the beta coefficients of the age effect on self-similarity. To characterise self-to-other-similarity association with age difference between time-point 1 and time-point 2, we performed a linear mixed-effects model (LME) with days between scans as fixed effect and with subject at time-point1 dependent random effect for the intercept to account for repeated measures.2.4.3Given the distinct trajectories of maturation of subcortical and cortical regions, we repeated all the analysis described above in 7 clusters that represent larger anatomical areas: central, frontal, limbic, occipital, parietal, deep grey matter and temporal. These regions were composed of 8, 22, 14, 14, 10, 8, and 14 nodes respectively. The central cluster was composed of bilateral precentral and postcentral gyrus, paracentral lobules and supplementary motor areas. The frontal cluster was composed of superior, middle, inferior and orbitofrontal cortices, as well as the olfactory lobule and the rectus gyrus. The limbic cluster was composed of the insula, anterior and posterior cingulate, hippocampus and amygdala. The occipital cluster was composed of the calcarine, cuneus, lingual cortices together with superior, inferior and middle occipital gyrus and the fusiform. The parietal cluster was composed of the superior and inferior parietal gyrus, supramarginal, angular and precuneus gyrus. The deep grey matter cluster was composed of all basal ganglia structures and thalami. The temporal cluster was composed of the Rolandic operculum, the Heschl gyrus, and superior, middle and interior temporal cortices as well as the temporal poles. 33.1r = 0.90, ranging from r = 0.67 to r = 0.96, and the mean self-to-other-similarity was r = 0.88, ranging from r = 0.65 to r = 0.94. The identifiability rate was 28/45 (62.22%), representing the percentage of times self-similarity was higher than the self-to-other-similarity. Furthermore, we found a significant correlation between PMA at first scan and self-similarity values , controlling for days between scans .The structural connectome comparison between the scans at preterm and term equivalent ages yielded strong correlations not only among the scans of the same subject but also between the scans of different subjects AB. The mr = 0.56, ranging from r = 0.21 to r = 0.70, and the mean self-to-other-similarity was r = 0.54, ranging from r = 0.17 to r = 0.73. Thus, the identifiability rate for functional connectome similarity was 3/31 (9.68%). We observed no significant correlation between PMA at first scan and functional self- similarity, but there was a significant negative correlation between functional self-similarity and days between scans . The same similarity rate was observed when testing partial correlation FC, 3/31(9.68%). However, mean self-similarity was just r = 0.12, ranging from r = 0.04 to r = 0.19, and mean self-to-other-similarity was r = 0.11, ranging from r = 0.02 to r = 0.22 , in contrast with 3/26 (11.54%) for functional data . We observed no significant effect of days between scans on structural connectome self-similarity; and no effect of age at time point 1 or days between scans on global functional connectome self-similarity .Table 2BThe LME analysis showed age at first scan also has a significant effect on global structural connectome self-to-others-similarity , but there was no significant effect of days between scans. Unlike what was observed for self-similarity, both days between scans  and age at time-point 1  had a significant effect on global functional connectome self-to-others-similarity.3.3Regional comparison of structural connectomes showed lower identifiability rate compared to the global metrics. The limbic cluster  showed highest identifiability rate at 11/45 (24.44%), followed by frontal regions at 10/45 (22.22%), occipital at 7/45 (15.56%), central at 5/45 (11.11%), both parietal and deep GM at 4/45 (8.89%) and temporal at 3/45 (6.67%).Functional connectome comparisons yielded similar identifiability rates compared to whole-brain metrics. They were still qualitatively lower than structural similarity identifiability rates, except for the parietal cluster. Central and parietal regions showed the highest identifiability rate of 3/31 (9.68%). Limbic regions showed an identifiability rate of 2/31 (6.45%) and frontal, occipital, sub-cortical and temporal clusters showed an identifiability rate of 1/31 (3.23%) .Fig. 4StCloser qualitative inspection of the self-similarity for the structural and functional connectivity in the sub-group of participants with both structural and functional connectomes showed structural and functional self-similarity closer together in the central cluster and more dispersed in the frontal cluster . However3.4The generalised linear model run independently for each cluster in the sub-group with both structural and functional data showed that age at time-point 1 has a significant effect on functional connectome similarity in the parietal region after Bonferroni correction . We observed no significant effect of age or days between scans on any other cluster for functional nor structural self-similarity after multiple comparisons correction .4In the current study we used a unique set of longitudinal high quality structural and functional neonatal brain MRI data from the Developing Human Connectome Project to investigate the status of the connectome fingerprint at an early stage of neurodevelopment. To do so, we selected data from preterm born infants that were scanned soon after birth and then again at term equivalent age. Our results show that the whole-brain structural connectome can already identify an older individual at birth. In contrast, the whole-brain functional connectome changes more noticeably between scan timepoints, so individual identification is less stable based on their functional connectome regardless of their age at birth or time between scans.4.1During the perinatal period, the brain undergoes marked micro and macrostructural changes . Neverth4.2Task-based fMRI studies in neonates have demonstrated that the primary sensory cortices  are capable of processing external stimuli and are undergoing activity-dependent maturation during the perinatal period . RestingOur results markedly contrast with those of previously reported work in children. To our knowledge, the youngest previously investigated population for connectome similarity analysis to date has been a sample of 6-year-old children, where they reported an identification rate of 43% between resting state functional connectomes . AnotherAn additional factor that should be considered is the impact of motion artefacts on functional connectivity . Adult s4.3To disentangle the impact of inter-scan interval and age at first scan on similarity of the structural and functional connectome, we examined a sub-group of 26 neonates who had both data types. We observed a significant effect of age at time-point 1 on the global structural self-similarity. This effect might be determined by the individual-specific developmental trajectory of white matter microstructure and not necessarily indicative of an adult-like unique structural connectome .The absence of any significant effect of age on global functional self-similarity at this early stage of development might be explained by the reshaping of long-range functional connectivity, which matures within the first postnatal year . HoweverInterestingly, we observed the same age effect on global structural connectome self-to-other-similarity. This suggests certain age dependent organisation patterns are also common across subjects. The same was observed for functional self-to-other-similarity, which significantly decreased with longer intervals between scans being compared and increased with older age at time point 1. The low self-similarity, combined with strong self-similarity-to others and its age dependency roots for time constrained functional changes at a developmental time where there is a strong mixture of finely tuned spontaneous neural activity patterns and sensory experience dependent mechanisms . The str4.4Brain development broadly follows a posterior to anterior maturational trajectory  with senWe saw the highest identifiability rate in the central cluster of the functional connectome. This suggests that functional sensory-motor networks can provide higher identifiability rates in babies, while in comparison frontal-parietal structures appear more unique in adults . However4.5The main limitation of this study is that to assess the connectome fingerprint across weeks in the perinatal period ex-utero there is no other option but to investigate a preterm born population. Hence, to what extent the fingerprint is affected by preterm birth or is representative of normal development will have to be investigated in older infant cohorts or using foetal MRI. Another limitation pertains the multiple developmental factors which may influence the acquired signal in different ways and consequently the connectome. Despite the robustness of SIFT to characterise structural connections , using t5The brain structural connectome fingerprint is already present in the perinatal period: it is relatively stable and individually unique at this stage of development. The identification features of functional connectivity are more complex to interpret, potentially being too dynamic or immature to provide a fingerprint. Region-wise analysis suggested that the functional fingerprint in early development might be more stable within regional clusters, although identifiability rates were still higher for structural data. Future studies should investigate regional differences throughout development, the association of the global structural fingerprint to developmental outcome, and whether genetic or environmental risk impact the stability of the fingerprint.https://data.developingconnectome.org/.The dHCP is an open-access project. The imaging and collateral data can be downloaded by registering at github.com/code-neuro/neonatalconnectomefingerprint/.Derived data including structural and functional connectivity networks used in this study are available in 10.13039/100010663ERC Grant Agreement no. 319456. The authors acknowledge infrastructure support from the National Institute for Health Research (NIHR) Mental Health Biomedical Research Centre (10.13039/100014461BRC) at South London, Maudsley NHS Foundation Trust, 10.13039/501100000764King's College London and the NIHR-BRC at Guys and St Thomas’ Hospitals NHS Foundation Trust (GSTFT). The authors also acknowledge support in part from the Wellcome Engineering and Physical Sciences Research Council (10.13039/501100000266EPSRC) Centre for Medical Engineering at King’s College London [WT 203148/Z/16/Z] and the 10.13039/501100000265Medical Research Council (UK) [MR/K006355/1]. Additional sources of support included the Sackler Institute for Translational Neurodevelopment at King’s College London, the European Autism Interventions (EU-AIMS) trial and the EU AIMS-2-TRIALS, a European Innovative Medicines Initiative Joint Undertaking under Grant agreements nos. 115300 and 777394, the resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (Grant FP7/2007–2013). DC is supported by the 10.13039/501100003130Flemish Research Foundation . TA is supported by a MRC Clinician Scientist Fellowship [MR/P008712/1]. DE received support from the Medical Research Council Centre for Neurodevelopmental Disorders, King’s College London [MR/N026063/1]. DB received support from a Wellcome Trust Seed Award in Science [217316/Z/19/Z]. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.This work was supported by the European Research Council under the European Union Seventh Framework Programme (FP/2007–2013)/The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.